Vaccinia virus, despite its high level of reactogenicity, remains the only available option for protection against smallpox infection. Due to concern about the potential use of smallpox virus as a bioweapon, smallpox vaccination currently is being reintroduced. However, the immune defense mechanisms protecting a person from smallpox after vaccination are not completely understood. The smallpox vaccine provides a unique opportunity to evaluate T cell responses in truly separate cohorts (naive and experienced) to begin delineating the phenotypes and functional characteristics of these cells in protection from disease. Using state-of-the-art polychromatic flow cytometry, we can now examine a multitude of lymphocyte subsets in the peripheral blood that previously were inaccessible, each with a unique functional and phenotypic profile. By studying patients with adverse events, we may be able to assign clinical relevance to some of these subsets. The goal of this research program is 1) to identify the functional and phenotypic profile of vaccinia virus-specific T cells elicited following smallpox immunization and 2) to examine the persistence of vaccinia-specific T cells in vaccinia-experienced vaccinees and the breadth of the immune response in immune and non-immune subjects by determining the Vbeta repertoire usage by those T lymphocytes. Distinct T cell subsets respond differently to viral infections and characterization of these subsets may lead to a better understanding of the immune response and the role of these cells in shaping long-term memory responses. Thus, we will learn not only about immunity to vaccinia, but also learn about the basic biology of virus-specific T cell responses. This work will provide a foundation for comparison of the cellular immunogenicity induced by other candidate vaccines, such as further attenuated viruses like MVA, or cell culture derived vaccinia. Given that a true efficacy trial cannot be conducted with these vaccines, likely correlates of immunity are needed for comparative purposes.